Effect of antigen/antibody ratio on macrophage uptake, processing, and presentation to T cells of antigen complexed with polyclonal antibodies.

Activation of a galactosidase-specific murine T hybridoma clone and of a human tetanus toxoid-specific T clone by antigen-presenting cells (APC) was used to evaluate the regulatory function of antibodies complexed with the relevant antigen. Complexed antigen, in fact, is taken up with high efficiency thanks to Fc receptors borne by APC. Antibody/antigen ratio in the complexes proved to be a critical parameter in enhancing antigen presentation. Complexes in moderate antibody excess provided optimal T cell activation independently of the physical state of the complexes (precipitated by a second antibody or solubilized by complement). Complexes in extreme antibody excess, on the contrary, did not yield T cell activation although taken up by APC efficiently. The effect of antibodies at extreme excess was observed with substimulatory dose of antigen (loss of potentiation) and with optimal dose of antigen (loss of stimulation). An excess of specific polyclonal antibodies hampers proteolytic degradation of antigen in vitro, supporting the view that a similar mechanism may operate within the APC that have internalized immune complexes in extreme antibody excess. The possibility that immune complex forming in extreme antibody excess may turn off the T cell response is proposed as a regulatory mechanism.

Efficacy of third-party chimeric antigen receptor modified peripheral blood natural killer cells for adoptive cell therapy of B-cell precursor acute lymphoblastic leukemia.

We developed an innovative and efficient, feeder-free culture method to genetically modify and expand peripheral blood-derived NK cells with high proliferative capacity, while preserving the responsiveness of their native activating receptors. Activated peripheral blood NK cells were efficiently transduced by a retroviral vector, carrying a second-generation CAR targeting CD19. CAR expression was demonstrated across the different NK-cell subsets. CAR.CD19-NK cells display higher antileukemic activity toward CD19+ cell lines and primary blasts obtained from patients with B-cell precursor ALL compared with unmodified NK cells. In vivo animal model data showed that the antileukemia activity of CAR.CD19-NK cell is superimposable to that of CAR-T cells, with a lower xenograft toxicity profile. These data support the feasibility of generating feeder-free expanded, genetically modified peripheral blood NK cells for effective "off-the-shelf" immuno-gene-therapy, while their innate alloreactivity can be safely harnessed to potentiate allogeneic cell therapy.

Quantitative competitive reverse transcriptase-polymerase chain reaction for BCR-ABL on Philadelphia-negative leukaphereses allows the selection of low-contaminated peripheral blood progenitor cells for autografting in chronic myelogenous leukemia.

The Philadelphia (Ph) translocation t(9;22) results in the creation of the BCR-ABL gene, which is now regarded as central to the mechanism that underlies the chronic phase of chronic myelogenous leukemia (CML). From a clinical point of view, BCR-ABL mRNA detection has become the basis for the study of minimal residual disease in CML, particularly when a complete cytogenetic remission is achieved after interferon-alpha (IFN-alpha) therapy or allogeneic stem cell transplantation. We have recently demonstrated that it is possible to mobilize normal peripheral blood progenitor cells (PBPC) in higher rates if this procedure is performed during the early chronic phase. In an attempt to monitor the leukemic cell content of PBPC collections, we used quantitative-competitive RT-PCR (QC-RT-PCR). Thirty consecutive Philadelphia (Ph) chromosome positive patients were enrolled in this study. After chemotherapy and G-CSF, 14 patients achieved 100% Ph-negative metaphases, nine patients had < or =34% and seven patients >34% leukemic metaphases. A total of 116 collection samples were studied. For each sample, BCR-ABL transcript numbers and BCR-ABL/ABL ratio were evaluated. A highly significant correlation between Ph-positive metaphases and BCR-ABL transcript numbers (r = 0.84, P < 0.0001) or BCR-ABL/ABL ratio (r = 0.86, P < 0.0001) was found. For patients that underwent the procedure in early chronic phase, Ph-negative collections showed different levels of BCR-ABL expression. BCR-ABL transcript numbers varied from a median of 100/microg RNA in the first and second leukaphereses, to 500/microg RNA in the third and fourth leukaphereses, and 1500/microg RNA in the fifth leukapheresis (P = 0.002). BCR-ABL/ABL ratio values showed similar kinetics. We have also demonstrated that there is a correlation between low values in BCR-ABL/ABL ratio (< or =0.01) in the reinfused PBPC and the achievement of cytogenetic remission after autografting (chi2 test, P = 0.01). In conclusion, this study demonstrates that QC-RT-PCR for BCR-ABL is a reliable and helpful method for monitoring residual leukemic load in mobilized PBPC, particularly in Ph-negative collections. Moreover, QC-RT-PCR allows selection of the best available collections for reinfusion into patients after myeloablative therapy.

Cytogenetic response to autografting in chronic myelogenous leukemia correlates with the amount of BCR-ABL positive cells in the graft.

OBJECTIVE: An important step in successful autografting of patients with chronic myelogenous leukemia is the delivery of a leukemia-free graft. We conducted this study to determine whether the cytogenetic response after autografting was correlated with the number of BCR ABL-positive cells present within the stem cell grafts. MATERIALS AND METHODS: By BCR-ABL mRNA quantification, we studied the serial pheresis products from 40 Philadelphia (Ph)-positive patients who received ICE/mini-ICE mobilization therapy and underwent autologous stem cell transplantation. We correlated the residual disease within the graft reinfused with the cytogenetic response following transplantation, taking into consideration those responses that lasted 12 months or more. RESULTS: Thirty-two patients received a graft with 0-35% Ph-metaphases and 19 received a graft with BCR-ABL/ABL ratio < or =0.01. After a median of 27 months (range, 12-50) from transplant, 18 patients achieved complete or major cytogenetic response lasting at least 12 months, and 14 of them (78%) received a graft with BCR-ABL/ABL ratio < or =0.01 (range, 0.0003-0.01). Twenty-two patients experienced short-lived responses or had >35% Ph-positive cells in the marrow after transplant, but only 5 of them (23%) had a graft with BCR-ABL/ABL ratio < or =0.01 (range, 0.001-0.01). Therefore, we found a strong association between a BCR-ABL/ABL ratio less than or =0.01 and the achievement of complete or major cytogenetic remission after autografting (chi(2) test, p = 0.0001). Patients reinfused with grafts contaminated at low levels with leukemic cells also showed a longer duration of the response (log-rank test, p = 0.0009). Eleven patients were reinfused with the lowest level of contaminated stem cell collections, according to the BCR-ABL/ABL ratio. None of these patients experienced prolonged neutropenia or thrombocytopenia following stem cell reinfusion and nine of them had long-lasting complete or major cytogenetic responses after transplant. CONCLUSION: This study demonstrates that the number of BCR-ABL positive cells present in a stem cell graft is an important predictive factor for the achievement and the duration of cytogenetic response after autografting. [corrected]

Dendritic cells are potent antigen-presenting cells for in vitro induction of primary human CD4+ T-cell lines specific for HIV gp120.

Activation of T helper cells specific for viral antigens is critical for antibody production and for generation of cytotoxic cells during the immune response to HIV. Since T-cell activation depends on antigen-presenting cells (APCs), it is important to define the cells that have a role in presentation of HIV antigens in general and of gp120 in particular. Peripheral blood mononuclear cells (PBMCs), adherent monocytes (AMs), dendritic cells (DCs) and Epstein Barr virus-transformed B-cell lines (LCLs) were tested for the capacity to present gp120 to a specific T-cell clone. DCs proved to be the most effective APC. Primary T-cell lines were generated from uninfected and unprimed individuals by using different APCs in the presence of gp120 or an immunodominant peptide. T-cell lines specific for gp120 were obtained with PBMCs or DCs as APCs, but not with AMs or LCLs. The data showed that (a) DCs are the most effective APCs for presentation of gp120 to specific T cells and (b) DCs are necessary for in vitro induction of primary T-cell lines specific for gp120.

Rational reconstitution of the immune repertoire in AIDS with autologous, antigen-specific, in vitro-expanded CD4 lymphocytes.

HIV infection leads to decrease of CD4 lymphocytes. In particular, loss of CD4 cells specific for opportunistic pathogens results in active opportunistic infections, that are the chief cause of morbidity and mortality in AIDS. Highly active anti-retroviral therapy (HAART) has been shown to have dramatic effects in a large fraction of treated individuals, such as decrease of viral load and increase of CD4 cells. It has not been clearly established, though, whether CD4 cells that appear during HAART represent a functional repertoire (i.e. a CD4 repertoire that encompasses all specificities, including T-cells responding to opportunistic pathogens, as in immunocompetent individuals) or an amplified mirror image of a defective repertoire. Therefore we propose that the immune repertoire can be reconstituted with autologous CD4 lymphocytes collected at early stages of infection, selected for specificity for opportunistic pathogens, expanded and stored for future use when laboratory or clinical signs indicate a depletion of antigen-specific CD4 cells. Since the reinfused CD4 cells are themselves susceptible to HIV infection-replication, we suggest that in vitro gene therapy of these cells may confer a genetic resistance that is permanently maintained in these cells.

Analysis of the antigen specific T cell repertoires in HIV infection.

In addition to HIV infection, several acquired immunodeficiencies lead to depletion of CD4 lymphocytes. These include immunosuppression resulting from high dose cancer chemotherapy or induced to control graft rejection, as well as in autoimmune diseases. The consequence of this depletion is an increased susceptibility to opportunistic infections or the inability to control primary infection in the case of HIV infection. In all instances a full or partial immunoreconstitution is desirable. In order to monitor the cellular immune state of a patient, rational information cannot be simply derived from phenotypic quantification of T lymphocytes. Instead loss or recovery of CD4 cells should be monitored by defining the specificity, the function and the clonality of the relevant cell population. Several methods are now available for this type of investigation. Here we describe an approach for the definition of clonal heterogeneity of antigen specific CD4 lymphocytes, a parameter that may help monitor loss or reconstitution in acquired immunodeficiencies. As examples of antigen specific CD4 T cell responses we focused on Pneumocystis carinii and on cytomegalovirus, as prototypic opportunistic pathogens which are responsible for severe infections in AIDS and in other immunosuppressive conditions which arise for instance following transplantation. Specific CD4 T cell lines were generated from normal controls and from seropositives in order to select antigen specific lymphocytes. The cells were subsequently analyzed for clonal diversity according to TCR BV gene family usage and according to TCR CDR3 size heterogeneity (spectratyping).

Repertoire breadth of human CD4+ T cells specific for HIV gp120 and p66 (primary antigens) or for PPD and tetanus toxoid (secondary antigens)

Antigen derived peptides bound on MHC class II molecules on presenting cells stimulate specific CD4 lymphocytes that are in a naive state if antigen is given for the first time, or in a memory state if antigen has been previously encountered. In order to compare clonal heterogeneity of the human CD4+ T helper repertoire in primary vs. recall responses, we have generated T cell lines in vitro by repeated stimulation of peripheral lymphocytes with primary or with recall antigens. Clonal heterogeneity was broad in the case of recall response to tetanus toxoid or PPD, with a high frequency of specific precursors (> 100 cells/10(6) lymphocytes). In contrast, T cell lines responsive to primary antigens (HIV gp120 or HIV p66) were oligoclonal as defined by TCR V beta gene usage and by spectratyping, and the precursor frequency was low (< 2 cells/10(6) lymphocytes). Primary T cell lines generated from blood samples drawn at different times from the same donor showed that clones with identical TCR CDR3 region coding sequences were expanded, suggesting that in these individuals a large progeny derived from one single precursor is present, even though a previous encounter with the antigen was not documented. Assuming an even in vivo distribution of such cells, the presence of one precursor every 10(6) CD4 lymphocytes (within the CD4 T repertoire that comprises roughly 10(11) CD4 T cells) indicates that approximately 10(5) identical T cells from the same clonal precursor account for the primary response against the model antigens we have studied.

Requirement for different presenting cells and for different processing mechanisms by human CD4 T helper clones specific for M. tuberculosis antigens.

Human T helper cells specific for mycobacterial antigens have been extensively investigated. Differences have been detected according to antigen specificity and to fine epitope specificity. In this work we have analyzed two additional parameters that allow discrimination among antigen specific T helper cells: requirement for certain types of antigen presenting cells (APC) and requirement for protease-sensitive antigen processing pathways. We used T cell clones from peripheral blood or from pleural exudates, and specific for different antigenic fractions of M. tuberculosis. APC were autologous peripheral blood mononuclear cells, adherent monocytes, adherent pleural monocytes, EBV transformed B lymphocytes and dendritic cells. Seven clones out of twelve were stimulated by all APC irrespective of their specificity, whereas other clones had more selective requirements. When protease inhibitors were used during antigen pulsing of APC, the production of certain epitopes, and thus T cell activation, was impaired with six clones out of sixteen. These results demonstrate that the human T helper repertoire specific for mycobacterial antigens is highly diverse also according to APC populations needed for presentation and to processing mechanisms required for production of the relevant T epitopes.

In vitro selection of HIV and CMV specific T-lymphocytes.

CMV and HIV produce life-long infections. During CMV infection, cellular responses mediated by virus specific CD8 and CD4 lymphocytes are effective, while during HIV infection cellular responses are ineffective in the long run. In recent years, much work has been carried out to better characterize such responses by using different methodologies to define the fine epitope specificity, the frequency and the function of specific T-cells. These studies have diagnostic and therapeutic implications. In fact, monitoring of specific lymphocytes may help define the immune status of the patients for therapeutic interventions. Identification of CD8 and CD4 epitopes allows the use of relevant peptides for lymphocyte stimulation or for vaccine development. Enumeration of specific cells permits a quantitative estimate of the immune response. In vitro selection provides large numbers of virus specific T-cells for studies on clonal composition, on epitope mapping and on HLA restriction as well as for therapeutic immunoreconstitution with ex vivo expanded T-cells.

Genetically modified immunocompetent cells in HIV infection.

Even in the era of highly active antiretroviral therapy (HAART), gene therapy (GT) can remain a promising approach for suppressing HIV infection, especially if complemented with other forms of pharmacological and immunological intervention. A large number of vectors and targets have been studied. Here we discuss the potential of genetically treated, antigen-specific immunocompetent cells for adoptive autologous immunotherapy of HIV infection. Cellular therapies with gene-modified CD8 and CD4 lymphocytes are aimed at reconstituting the antigen-specific repertoires that may be deranged as a consequence of HIV infection. Even if complete eradication of HIV from the reservoirs cannot be achieved, reconstitution of cellular immunity specific for opportunistic pathogens and for HIV itself is a desirable option to control progression of HIV infection and AIDS pathogenesis better.

Kinetic immunodominance: functionally competing antibodies against exposed and cryptic epitopes of Escherichia coli beta-galactosidase are produced in time sequence.

The murine antibody response to Escherichia coli beta-galactosidase (GZ) was analyzed in vivo and in vitro by focusing on two families of antibodies that exert distinct conformational/functional activity on the antigen. Activating antibodies--defined by their capacity to increase the enzymatic activity of defective GZ produced by mutant strains of E. coli--are detected early after secondary challenge. Inhibiting antibodies, which interfere with antibody-mediated enzyme activation, appear later and cause the abrupt fall of activating titer, a scenario suggesting either idiotype/anti-idiotype interaction or opposite pulsions exerted on the antigen molecule. Supporting the latter mechanism, the confrontation of mAbs of the two families produced classical competitive inhibition curves when the readout was enzyme activation, although they recognize two different epitopes of the same molecule: the activating mAb a quaternary conformation-dependent site of wild-type GZ, the inhibiting mAb a sequential determinant exposed only in denatured or in defective enzyme. The different timing of generation of these antibodies during the response may depend on a processing step necessary for unfolding of native antigen and consequent display of certain cryptic epitopes before they can trigger specific B cells. A picture emerges where the response to the various epitopes of a complex antigen is sequentially connected and where the uptake by antigen-presenting cells of antigen complexed with antibodies specific for the exposed epitopes may favor revelation of the cryptic ones.

Role of flanking variable sequences in antigenicity of consensus regions of HIV gp120 for recognition by specific human T helper clones.

Human T helper cells can discriminate among strain variants of HIV gp120 because of T cell clones recognizing non-conserved regions, as demonstrated with T cells from HIV-infected individuals and vaccinated volunteers and with primary T cell lines and clones obtained by in vitro immunization. To obtain a better definition of cross-reactions among T cell determinants within HIV gp120 variants, we used a panel of analog peptides within residues 236-251 from the BRU, MN, SF2 and RF strain sequences to induce primary human T cell lines and clones. Different patterns of response were obtained using each of the analog peptides, although they all share the consensus sequence 246-251. Clones recognizing this sequence were generated by priming with the BRU and RF analog peptides, but not with the SF2 analog peptide. SF2 did not induce any 242-245-specific clones, but only T cells recognizing the 236-240 sequence. A preferential response to residues 236-240 was obtained by priming with the BRU and SF2 peptides, but not with the MN and RF peptides. These results suggest that although the analog peptides exhibit a high degree of homology and share a consensus of the C-terminal sequence (246-251), the T cell response to the conserved sequence 246-251 is controlled by flanking sequences. Therefore the presence of a shared sequence per se does not imply in vitro expansion of clones with that fine specificity, even though such clones are available within the naive repertoire and can be triggered by an analog peptide.

Limited clonal heterogeneity of antigen-specific T cells localizing in the pleural space during mycobacterial infection.

To detect possible differences in phenotype and fine specificity for mycobacterial antigens between CD4-positive T cells from peripheral blood (PB) and from inflammatory sites, we identified four patients presenting with a mycobacterial pleural exudate (PE) rich in PPD-specific lymphocytes and with a negative skin test to tuberculin purified protein derivative (PPD) and a negative proliferative response of PB lymphocytes to PPD at the same time. Several weeks after chemotherapy, these patients converted to PPD responsiveness in the periphery, and PPD-specific clones could be generated from PB at this stage. The phenotypic comparison of PE lymphocytes and concomitant PB lymphocytes obtained before treatment showed an increase of CD8 cells and a high frequency of HLA-DR-positive activated T cells in PE. The frequency of tetanus toxoid-specific and Candida albicans-specific proliferating T cells was lower than that of PPD-specific cells in PE but not in PB. PPD-specific clones were derived initially from PE and from PB once the patients had converted to PPD responsiveness. The two sets of clones from each patient were compared for proliferative response to mycobacterial antigen clusters of defined molecular weight ranges. A large number of PE-derived clones (36%) responded to a fraction of 27 to 35 kDa, whereas only one clone from PB responded to the same fraction. The purified antigen P32 (32 kDa), a soluble mycobacterial protein, stimulated PE-derived clones that were responsive to the 37- to 27-kDa fraction but did not stimulate PB-derived clones. The data demonstrate that PE- and PB-derived lymphocytes differ both in phenotype and in fine specificity, suggesting a limited clonal heterogeneity of T cells localizing at the inflammatory site in tuberculous patients without a PPD response in the periphery. Therefore T cells compartmentalized at inflammatory sites provide information that is different from that provided by T cells in the periphery.

Antigenicity of HIV-derived T helper determinants in the context of carrier recombinant proteins: effect on T helper cell repertoire selection.

T helper (Th) epitopes can be included in a recombinant protein with B and CTL epitopes to create more effective immunogens. To determine whether the antigenicity of HIV Th epitopes is preserved in this altered molecular context, human Th clones specific for peptides of HIV gp120 and reverse transcriptase p66 were challenged with recombinant proteins carrying the antigenic epitopes in different sites. We found that a given epitope was recognized by a specific T cell clone only when it was inserted in a particular position of the carrier. However, the permissive position was not the same for all epitopes. Enzymatic excision from a nonpermissive context or insertion of a polyserine spacer between the epitope and the carrier restored antigenicity. Nevertheless, antigenicity was not abolished in a synthetic peptide encompassing the epitope and the neighboring residues from the nonpermissive location. These data suggest that, in this case, the primary sequence of the chimeric protein flanking the HIV peptide is not responsible for loss of antigenicity. Furthermore, constructs carrying the epitope in a given position were recognized by peptide-specific Th clones raised from some individuals, but not from others. We show that this is due neither to individual modes of processing nor to the use of distinct major histocompatibility complex MHC class II restriction elements, but rather that it is related to the fine specificity of the clones. To study the effect of epitope context on selection of T cell repertoire in a naive individual, T cell lines were generated in vitro by stimulation with different peptide constructs. This resulted in the induction of diverse clonotypes defined by the pattern of recognition of different constructs, by T cell receptor V beta gene usage and by fine epitope mapping.

Anti-HIV genetic treatment of antigen-specific human CD4 lymphocytes for adoptive immunotherapy of opportunistic infections in AIDS.

HIV-1 infection results in the loss of CD4+ T helper lymphocytes which make up the immune repertoire. This leads to opportunistic infections that define AIDS. Here, we show that CD4 T cell lines from normal donors with specificity for different antigens can be rendered resistant to HIV-1 replication by retroviral transduction with an antisense vector directed to the HIV-1 tat gene. The genetic treatment did not affect the properties of antigen-specific CD4 lymphocytes such as proliferative response, lymphokine production and phenotypic markers. The HIV-1 challenge dose that resulted in productive infection was two to four logs higher for transduced cells as compared with control cells. Resistance was shown with the HXB2 strain, whose tat sequence was used to design the antisense gene, and with the SF2 strain, whose targeted tat sequence carries five nucleotide mismatches. Retroviral transduction was also performed on a Candida-specific T cell line from a seropositive individual. This line, derived from T cells infected in vivo, produced infectious virus when stimulated in vitro with antigen, but was no longer productive after transduction. In addition, a four log higher HIV-1 challenge dose was needed for a productive superinfection of this T cell line. The production of antigen-specific CD4 T cells resistant to HIV-1 replication to be used in adoptive immunotherapy of opportunistic infections may represent a new form of gene therapy of AIDS.

Epitope focus, clonal composition and Th1 phenotype of the human CD4 response to the secretory mycobacterial antigen Ag85.

Lymphoproliferation of healthy donors was tested against mycobacterial antigens (PPD, Ag85, Ag85 peptides). All PPD responders recognized the secretory antigen Ag85 and the peptide specificity for Ag85B was defined. Peptide 91-108 was recognized by 85% of donors. In addition, all CD4 T cell lines generated from 12 donors against PPD or Ag85 responded to 91-108. When this peptide was used to generate T cell lines, the cells responded also to tuberculins from atypical mycobacterial species. Thus the cross-reactive peptide behaved as quasi-universal. The analysis of TCR-BV gene usage by cell lines showed that most Ag85-specific T cells correspond to 91-108-specific clonotypes. Intracytoplasmic staining of cell lines after phorbol myristate acetate stimulation resulted in dominance of interferon-gamma (IFN-gamma)-IL-4 double-positive cells, whereas antigen stimulation resulted in production of IFN-gamma only. The data show that peptide 91-108 is the major focus of the CD4 response to mycobacterial antigens in peripheral blood mononuclear cells and in T cell lines from PPD responders.

Attenuated Listeria monocytogenes carrier strains can deliver an HIV-1 gp120 T helper epitope to MHC class II-restricted human CD4+ T cells.

Listeria monocytogenes is a facultative intracellular pathogen which, following uptake by macrophages, escapes from the phagosome and replicates in the cytoplasm. This property has been exploited using recombinant L. monocytogenes as a carrier for the intracytoplasmic expression of antigens when MHC class I-restricted cytotoxic T lymphocyte responses are required. Much less is known of the ability of these bacteria to trigger MHC class II-restricted responses. Here, we demonstrate that after ingestion of L. monocytogenes expressing a T helper epitope from the gp120 envelope glycoprotein of HIV, human adherent macrophages and dendritic cells can process and present the epitope to a specific CD4+ T cell line in the context of MHC class II molecules. No significant differences were observed when the attenuated strains were trapped in the phagolysosome or impaired in the capacity to spread intracellularly or from cell to cell. Similar results were obtained using carrier proteins that were either secreted, associated with the bacterial surface, or restricted to the bacterial cytoplasm. A dominant expression of the TCR Vbeta 22 gene subfamily was observed in specific T cell lines generated after stimulation with the recombinant strains or with soluble gp120. Our data show that in this in vitro system L. monocytogenes can efficiently deliver antigens to the MHC class II pathway, in addition to the well-established MHC class I pathway. The eukaryotic cell compartment in which the antigen is synthesized, and the mode of display seem to play a minor role in the overall efficiency of epitope processing and presentation.